This project is focused on the genetic analysis of familial melanoma. In previous work we, and others, have shown that agene located in chromosomal region 9p21 plays an important role in the development of familial melanoma. Recently, a gene, p16, that inhibits an important enzyme (CDK4, cyclin dependant kinase) that regulates cell division has been mapped into the region that is linked to familial melanoma and homozygously deleted in many tumor cell lines. We have screened 18 families with hereditary melanoma, and identified 8 p16 germline mutations (1 nonsense, 1 splice donor site, and 6 missense) in 13/18 kindreds. Six mutations were identified in 33/36 melanoma cases in 9 families. Two mutations were detected in normal controls and are not disease-related. These data confirmed previous evidence of genetic heterogeneity since the melanoma-specific mutations were only detected in 9p21-linked, but not in 1p36-linked families. Functional analyses of these mutations will confirm those causally related to the development of familial melanoma. These data, combined with data from other groups, very strongly implicates the p16 gene as a tumor suppressor locus for melanoma. The characterization of these mutations has direct implications for presympomatic diagnosism of at-risk individuals, and for further elucidation of the molecular pathways leading to the development of melanoma